Protein splicing is a precise post-translational process in which an intervening protein sequence, intein, is removed from a precursor protein with the concomitant ligation of the flanking sequences, N- and C-exteins. Although the basic steps of protein splicing are well-known, the catalytic mechanisms of intein splicing are still poorly understood. Advancing our fundamental knowledge of protein splicing can have two major impacts: Inteins have found extensive applications in protein engineering and biotechnology and therefore are an indispensable tool for biomedical research and potentially for therapies of diseases. Because only unicellular organisms have inteins vital for their survival, intein inhibitors can develop into a new class of antimicrobial drug with little toxicity for human cells, especially for Mycobacterium tuberculosis (Mtu). We propose two new mechanistic hypotheses for conserved residues H73 and D422 in Mtu RecA intein. These two hypotheses have been supported by diverse experimental evidence from genetics, in vivo splicing data in intein mutants, solution NMR studies and by calculations. Our specific aims are to test these two hypotheses using a combination of NMR structural biology methods, biochemical characterization of splicing reaction and molecular dynamics simulation to prove or refute these two novel hypotheses. In the process, new methods and concepts for protein splicing will be generated. The results from our research will greatly enhance our understanding the mechanisms of protein splicing and contribute to the application of inteins in biotechnology and potentially in treating diseases. The long term goal is to delineate the complete catalytic mechanisms of protein splicing by applying solution NMR in an interdisciplinary approach for studying enzyme catalysis, structure, dynamics and function.